PROJECT SUMMARY Glioblastoma (GBM) is an incurable cancer even with aggressive therapies such as surgical resection followed by radiotherapy and chemotherapy using temozolomide (TMZ). Efforts to improve surgical resection or the efficacy of irradiation are limited by the potential damage these interventions cause to the brain. In contrast, sensitizing GBM to TMZ is an appealing strategy because TMZ has excellent brain penetration and a low toxicity profile. Recent research suggests that targeting the gap junction protein connexin 43 (Cx43) holds promise for enhancing TMZ sensitivity in GBM. A synthetic peptide, aCT1, which comprises the carboxy- terminus of Cx43, and has demonstrated therapeutic efficacy in promoting healing of acute and chronic wounds, has been developed in order to explore the potential of targeting Cx43 and overcoming TMZ resistance in GBM. FirstString Research has currently advanced Granexin gel, the topical formulation of aCT1 peptide, through three Phase 2 human clinical trials for scar reduction and the treatment of chronic wounds. Preliminary data demonstrated that Cx43 expression inversely correlates with TMZ sensitivity and GBM patient survival, and demonstrated that aCT1 significantly increases TMZ sensitivity in vitro and in vivo, thus encouraging further investigation into its therapeutic potential in sensitizing GBM tumors to TMZ. During the Phase I SBIR, to facilitate efficient and targeted drug delivery, a controlled and sustained biodegradable aCT1 nanoparticle system of therapeutic delivery was developed and validated in vitro and in vivo models of GBM. Biodegradable aCT1-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) were developed, optimized and validated, specifically with characteristics necessary for targeted convection-enhanced delivery (CED) in GBM patients (FDA approved copolymer; <150nm +\- 40 in diameter, controlled and sustained aCT1 release profile). The objective of this Phase II SBIR proposal is to translate the success of our Phase I data through more extensive pre-clinical development. Aim I will involve intracranial injection of aCT1-NPs into the brains of GBM mice followed by TMZ treatment and mechanism of action studies. We will monitor tumor growth using MRI and analyze mouse survival. Aim II will validate the efficacy and safety of combinatorial aCT1-NP and TMZ treatment in a veterinary clinical trial in high-grade spontaneous canine gliomas. Canine gliomas have many of the characteristics of human tumors, thus permitting precise extrapolation of efficacy and safety data from canine therapy studies to human trials. We will enroll companion dogs with spontaneous tumors into a specific protocol involving CED of aCT1-NPs in association with TMZ. Efficacy evaluation will involve comprehensive neuroimaging response assessment, neurobiobehavioral evaluation, and survival. Safety analyses will involve adverse event reporting and toxicokinetic analyses. Successful completion of these aims will validate CED delivery of aCT1-NP as a novel combinational therapy for lethal GBM and will lay foundation for potential clinical trials in newly diagnosed GBM patients in the near future.